Exploring the potential biological significance of KDELR family genes in lung adenocarcinoma

The Lys-Asp-Glu-Leu receptor (KDELR) family genes play critical roles in a variety of biological processes in different tumors. Our study aimed to provide a comprehensive analysis of the potential roles of KDELRs in lung adenocarcinoma (LUAD). Utilizing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database, as well as clinical samples, we conducted a series of analyses and validations using R software tools and various online resources. The results showed that KDELR family genes and proteins were highly expressed and associated with a poor prognosis of LUAD. Promoter hypomethylation and the competing endogenous RNA (ceRNA) network of PCAT6/hsa-miR-326/KDELR1 might be potential causes of aberrant KDELR1 overexpression in LUAD. Three key Transcription factors (TFs) (SPI1, EP300, and MAZ) and a TFs-miRNAs-KDELRs network (involving 11 TFs) might be involved in modulating KDELRs expression abnormalities. Gene Set Enrichment Analysis (GSEA) indicated enrichment of genes highly expressing KDELR1, KDELR2, and KDELR3 in MTORC1_SIGNALING, P53_PATHWAY, and ANGIOGENESIS. Negative correlations between KDELRs expression and CD8 + T cell infiltration, as well as CTLA-4 expression. Our multiple analyses suggested that the KDELRs are important signaling molecules in LUAD. These results provided novel insights for developing prognostic markers and novel therapies of LUAD.


RFS
Relapse-free survival TCGA The cancer genome atlas TFs Transcription factors TIME Tumor immune microenvironment TICs Tumor-infiltrating immune cells TME Tumor microenvironment Lung cancer accounts for the highest percentage of cancer deaths, with 1.8 million annual deaths recorded globally in recent years 1,2 .Non-small cell lung cancer (NSCLC), the predominant subtype, constitutes approximately 80% of all lung cancer cases, with lung adenocarcinoma (LUAD) being the prevailing histological subtype within NSCLC 1 .Despite notable advancements in the molecular pathology and targeted therapeutic strategies for LUAD, the 5-year overall survival (OS) rate for this malignancy remains dismally low at around 15% 3 .Given the low cure rate and high recurrence rate, a comprehensive understanding of the molecular underpinnings driving the initiation and progression of LUAD is essential for early detection, precision treatment, and prognosis monitoring.
The Lys-Asp-Glu-Leu receptor (KDELR), a member of the PQ cyclin family, recognizes and binds to the KDEL-like motif at the C-terminal of endoplasmic reticulum (ER) chaperonins (also known as ER proteins), and backtracks from Golgi to the ER via Coat Protein I (COPI) vesicles.This maintains the dynamic balance between the Golgi and the ER 4,5 .This was one of the first physiological roles of KDELRs to be discovered.Later, with further research, KDELRs were found to be pivotal regulators in cellular trafficking, immunity and autophagy [6][7][8] .Notably, recent findings suggest that KDEL receptors have a Golgi-retention function, acting as Golgi-gatekeepers 9 .In addition to traveling between the Golgi and ER, KDEL receptors also circulate between the Golgi and the plasma membrane, contributing to the cell surface binding of ER proteins, thereby fostering cell proliferation and migration 10,11 .Further exploration revealed that KDELR, as a receptor for ER proteins secreted at the cell membrane, has its own downstream signaling network, including trans-activation of the EGFR-STAT3 signaling pathway 12 .
Moreover, accumulating evidence have shown that KDELRs are associated with the development and progression of different kinds of tumors.For example, KDELR1 deficiency enhanced melanoma metastasis 13 , while activation of KDELR2 triggered HDAC3-mediated promotion of breast cancer proliferation 14 .KDELR2 also harnessed the mTOR signaling pathway to promote glioblastoma tumorigenesis 15 , and was implicated in unfavorable prognostic markers for gliomas 16 .It was demonstrated that KDELR2 acted as a driver of lung cancer invasion and metastasis 17 .KDELR3 was identified to be involved in promoting melanoma metastasis 13 .However, a systematic examination of the entire KDELR family in tumors, including LUAD, remains conspicuously absent in the extant literature.
To explore the potential biological role of the KDELR family in LUAD, our study analyzed the characteristics of its members in LUAD from multiple perspectives, including the differential expression of mRNA and proteins, alterations of base sequences of the genes themselves (Amplification, Deletion, and Mutation), and survival analyses of related indicators.Subsequently, to explore the upstream regulatory mechanisms underlying aberrant mRNA expression, we investigated two epigenetic modification modalities (DNA methylation and non-coding RNA regulation), as well as the impact of transcription factors (TFs) on gene expression.Through enrichment analysis, we then pinpointed potential downstream pathways implicated in the dysregulated expression of KDELRs, focusing particularly on those implicated in LUAD pathogenesis.Finally, we explored the potential role of KDELRs in the tumor immune microenvironment (TIME) of LUAD by assessing their correlation with immune cells and markers.The analysis process of our study is shown in Fig. 1.

Aberrant expression of different KDELR family members in LUAD patients
After collation, we procured expression data for KDELR1, KDELR2, and KDELR3 in 513 LUAD tumor samples and 58 normal samples within The Cancer Genome Atlas (TCGA) dataset.Considering the specific characteristics and distributional properties of the data, we opted to employ either the Wilcoxon rank-sum test or the Welch's t-test for comparative analyses.Results revealed significantly elevated mRNA expression levels of the KDELRs in LUAD tissues relative to normal tissues (p < 0.001) (Fig. 2A-C).Subsequently, we identified 57 pairs of LUAD tumors and their matched normal counterparts within the TCGA dataset.Comparative analysis of these paired samples demonstrated a significantly higher mRNA expression of all three KDELR family members in LUAD tissues compared to their corresponding normal tissues (p < 0.001) (Fig. 2D-F).
This pattern was also reproduced in an independent validation set consisting of 23 pairs of clinically matched samples from our institution, where mRNA expression levels in LUAD tissues were consistently higher than those in adjacent non-tumor tissues (Fig. 2G-I).To further validate the expression profiles of the KDELRs, we used the same method to compare the independent microarray dataset GSE33532, which consists of the expression data of 20 pairs of cancer and adjacent normal tissues.Similar results were obtained (Fig. 2J-L).For the comparison of paired samples, the Wilcoxon signed-rank test or paired-samples t-test was utilized as appropriate, depending on the data format characteristics of each group.
Next, the Human Protein Atlas (HPA) was used to directly contrast the protein expression of KDELR1/2/3 in LUAD and normal lung tissues via immunohistochemical staining.KDELR1 displayed heightened protein expression in LUAD tissues relative to its low expression in normal lung tissues (Fig. 2M).KDELR2 protein was moderately expressed in LUAD tissues but was virtually absent in normal lung tissues (Fig. 2N).Conversely, KDELR3 protein expression was undetectable in both normal and LUAD tissues (Fig. 2O).To further investigate the protein expression pattern of KDELR1/2/3, we utilized UALCAN, accessing data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database.Despite KDELR2 being absent in the LUAD dataset, we found that total protein expression of KDELR1 and KDELR3 was significantly higher in LUAD tissues compared to normal tissues (Fig. 2P,Q.p < 0.001).
In summary, our comprehensive analysis demonstrated that both mRNA transcripts and proteins of KDELRs exhibit increased expression in LUAD patients relative to normal controls.

Mutations and prognosis of KDELR family genes
We analyzed genomic alterations of the KDELR family in 3513 LUAD patients across ten studies and assessed their associations with OS and disease-free survival (DFS) using the cBioPortal website.The mutation rates for KDELR1, KDELR2, and KDELR3 were found to be 0.9%, 3%, and 1.2%, respectively (Supplementary Fig. S1A).Furthermore, we found genetic alterations in KDELRs were significantly correlated with shorter OS (Supplementary Fig. S1B, p = 0.0154) and DFS (Supplementary Fig. S1C, p = 0.0126) in LUAD according to the results of K-M plots and log-rank test.These findings indicated that genetic alterations in KDELRs might have prognostic implications for LUAD patients.

Methylation associated with KDELR expression and prognosis
As depicted in Fig. 4A-C, MethSurv was used to show the visualization of the methylation levels of individual CpGs corresponding to KDELR1, KDELR2, and KDELR3 in LUAD.Differential methylation patterns were observed across the CpG sites, potentially reflecting the complexity of epigenetic regulation of KDELR gene expression in LUAD.In the survival analysis, we discovered that LUAD patients exhibiting hypomethylation at cg19677683 within KDELR2 and at cg01640635 within KDELR3, demonstrated significantly improved OS (p < 0.05; Fig. 4D,E), Conversely, LUAD patients with hypomethylation at cg20074795 within KDELR3 had significantly worse OS (p < 0.05; Fig. 4F).In the comparative assessment utilizing UALCAN, while no significant differences were observed in the promoter methylation levels of KDELR2 and KDELR3 between TCGA-LUAD tumor tissues and their corresponding normal counterparts (Fig. 4H,I), a striking disparity was evident for KDELR1, where its promoter methylation was notably lower in tumor tissues compared to adjacent normal tissues (p < 0.01) (Fig. 4G).This finding contrasted with the mRNA expression pattern of KDELR1.
Experimental validation by Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in clinical tissue and cell line samples revealed that KDELR1 and PCAT6 had higher expression, whereas hsa-miR-326 showed lower expression in the A549 cell line compared to the BEAS-2B cell line (Fig. 6E).Analogous results were obtained when comparing 23 clinical LUAD tissues and adjacent matched normal tissues (Figs.1D, 6F,G).As shown in Fig. 6H, PCAT6 was significantly negatively associated with has-miR-326 in clinical LUAD tissues.These validation results largely concurred with the outcomes of our earlier database analyses.

Construction of transcription factor regulatory networks
Based on comparative analysis of the promoter sequences of the target genes with respect to the DNA-binding domains of TFs, followed by stringent filtration criteria encompassing "forward strands" and "score > 20", the estimated count of TFs potentially interacting with KDELR1, KDELR2, and KDELR3 was respectively culled down to 14, 43, and 23.Subsequent Venn diagram intersection (Supplementary Fig. S2A) led to the selection of three key TFs-SPI1, EP300, and MAZ.Bioinformatic analyses (Supplementary Fig. S2B) uncovered the most highly predicted binding elements for these three key TFs within the corresponding promoter regions of KDELR1/2/3.

Functional enrichment analysis and PPI networks of KDELRs
The functions of KDELR1/2/3 and a cohort of 60 genes (selected from GEPIA2) sharing similar expression patterns were inferred via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis executed in Metascape [19][20][21] .These annotations were classified into four main categories: GO biological process group (10 items), GO cellular component group (7 items), GO molecular function group (2 items), and KEGG pathway group (1 item).KDLR1/2/3 and the comparable 60 genes were predominantly enriched in vesiclemediated transport processes from the ER to the Golgi apparatus, as evidenced in Supplementary Fig. S3A.
Considering that the molecular machinery and metabolism requirements of malignant tumors necessitate functional interactions among proteins, we proceeded to analyze the protein-protein interaction (PPI) network of KDELRs and the proteins encoded by the 60 similarly expressed genes (Supplementary Fig. S3B).From this PPI network encompassing 63 proteins, the two most significant Molecular Complex Detection (MCODE) components were extracted.Their biological functions were primarily related to Golgi vesicle transport, retrograde vesicle-mediated transport from Golgi to ER, ER to Golgi vesicle-mediated transport, and the Sphingolipid signaling pathway (Supplementary Fig. S3C).Furthermore, we procured PPI network data for KDELR1/2/3 proteins, listing the top 10 predicted functional interactors in Supplementary Fig. S3D, including ARFGAP1, ARF1, COPB1, ARFGAP3, COPA, ASAP1, CLTA, XBP1, ASAP2, ARAP2.
Dividing the samples based on median expression levels, we categorized KDELR1/2/3 into high and low expression groups and subsequently analyzed the differential expression of 19,934 genes across these groups in TCGA dataset, ranking them according to their degree of upregulation.By contrasting the enrichment significance of target gene sets in the Molecular Signatures Database v7.0 against this ranked list, we conducted Gene Set Enrichment Analysis (GSEA).GSEA results were obtained by comparing the enrichment significance of the target gene sets in Molecular Signatures Databases v7.0 in this sort.The GSEA results indicated that the genes in the KDELR1 high-expression group were significantly enriched in the MTORC1_SIGNALING and P53_PATH-WAY within the HALLMARK collection (Fig. 7A) and concurrently enriched in four gene sets within the C2 collection, all of which were overexpressed in multiple lung cancer cell lines (Fig. 7B).The genes in KDELR2 high-expression group were enriched in the MTORC1_SIGNALING in HALLMARK collection (Fig. 7C) and one gene set in the C2 collection that is upregulated in LUAD cell lines (Fig. 7D).The genes in the KDELR2 lowexpression group were enriched in gene sets representing downregulated genes in KRAS-mutated lung cancer cell lines and animal models, sourced from HALLMARK, C2, and C6 collections (Fig. 7E-G).Similarly, the www.nature.com/scientificreports/KDELR3 high-expression group was enriched in the ANGIOGENESIS pathway within the HALLMARK collection (Fig. 7H), whereas the KDELR3 low-expression group was enriched in gene sets reflecting downregulated genes in KRAS-mutated lung cancer cell lines.Like KDELR2, these 2 gene sets are derived from the C6 and Hallmark collections, respectively (Fig. 7I,J).

Correlation of KDELR expression with immune characteristics
Among the patients with cancer, the frequency of lymphocytes infiltrating the tumor independently predicts survival and lymph node metastasis [22][23][24] .Thus, we stratified LUAD samples from the TCGA database into highand low-expression groups based on the median expression levels of KDELR1/2/3, respectively, and subsequently assessed the proportion differences of the 22 tumor-infiltrating immune cells (TICs) between these groups.It was observed that, correspondingly, six, eight, and nine immune cell populations displayed significantly distinct proportions in the high versus low expression groups of KDELR1, KDELR2, and KDELR3, respectively (Fig. 8A-C, p < 0.05).Subsequently, we examined the correlation between the expression levels of KDELR1/2/3 and the proportions of the 22 immune cell types in all LUAD samples.This analysis revealed significant correlations between five, six, and eight immune cell types with KDELR1, KDELR2, and KDELR3, respectively.Upon taking the intersection of the results from these two analytical approaches, the top four immune cell types most strongly associated with KDELR1 expression were B cells memory, T cells gamma delta, Dendritic cells activated, and Neutrophils (Fig. 8D).Similarly, for KDELR2, the top four significantly correlated immune cells were B cells memory, T cells CD8, Macrophages M2, and Dendritic cells activated (Fig. 8E).Whereas for KDELR3, they were T cells CD8, Macrophages M2, Dendritic cells resting, and Dendritic cells activated (Fig. 8F).These findings further substantiated that the levels of KDELRs exerted a significant influence on the immunological activity within the tumor microenvironment (TME).
To further elucidate the potential role of KDELR family genes in the infiltration of diverse immunocytes in LUAD patients, the TIMER database was utilized to investigate connections between KDELR family genes and a set of immunological markers widely recognized as counterparts of different immunocytes.Upon adjusting for tumor purity, we detected significant associations between the expression of KDELR family members and markers of CD8 + T cell (CD8A, CD8B), T cell (general) (CD3D, CD3E, CD2), M1 macrophage (COX2), Neutrophils (CD15, CCR7), NK cell (KIR3DL2), Th1 (T-BET), Th17 (IL17A) in LUAD (P < 0.05; Supplementary Table S2).Notably, all markers of CD8 + T cells, displayed significant negative correlations with the expression of all KDELR family members.Moreover, the proportion of CD8 + T cells demonstrated a significant negative correlation with KDELR2 and KDELR3 expression, while exhibiting a trend towards negative correlation with KDELR1 expression.

Discussion
Recent studies have increasingly implicated diverse molecules in the occurrence and development of lung cancer, especially LUAD 26,27 .Whereas KDELR, as a receptor for ER proteins, maintains a dynamic balance between the Golgi and the ER 4,5 , and circulates between the Golgi and the plasma membrane to contribute to the binding of ER proteins on the cell surface, thereby promoting cell proliferation and migration 10,11 .In addition to trans-activation of the epidermal growth factor receptor-STAT3 signaling pathway 12 , other signaling networks downstream of its own are unclear.Therefore, in this paper, we synthesized data from various aspects in the expectation of finding the potential biological significance of KDLER family genes in lung adenocarcinoma.
DNA methylation, a pivotal epigenetic modification, has long been studied for its role in transcriptional repression.When methylation occurs within gene promoter regions, CpG islands become methylated, preventing protein binding and leading to transcriptional silencing 28 .Conversely, low methylation levels in gene promoter regions can fail to inhibit oncogene transcription, contributing to tumor initiation.Jones et al. 29 confirmed that methyl-CpG-binding domain (MBD) proteins initially recognized methylated CpGs in promoter regions, subsequently recruiting histone deacetylase complexes to repress downstream gene expression.Recent studies further suggested that DNA methylation had a critical effect on the regulation of gene expression [30][31][32][33] .We found that KDELR1 promoter methylation in LUAD cancer tissues was prominently lower than that in neighboring normal tissues, contrasting with the gene expression of KDELR1.This suggests that reduced promoter methylation may contribute to the upregulated expression of KDELR1 in LUAD samples.
miRNAs are a group of ncRNA molecules that have been established as important regulators of gene expression [34][35][36] .Following survival, correlation, expression analysis, and qRT-PCR validation, we identified has-miR-326 as the most significant tumor-suppressive upstream miRNA of KDELR1.Previous studies have www.nature.com/scientificreports/identified that hsa-miR-326 is involved in embryonic development, immunoregulation, tumorigenesis, tumor growth, chemoresistance, cell invasion, and apoptosis 37,38 .Based on the ceRNA hypothesis, the ceRNA molecules (mRNAs, lncRNAs, pseudogenes, etc.) were able to compete to bind the same miRNAs to regulate each' other's expression levels via the miRNA response element 18,39 .Therefore, we predicted and identified potential lncRNAs acting as upstream regulators of the hsa-miR-326/KDELR1 axis.PCAT6 was screened out following expression, survival, and correlation analyses, as well as qRT-PCR validation.Recent studies identified that PCAT6 had the function of an oncogene in various tumors.Liu et al. 40 demonstrated that PCAT6 promotes prostate cancer development by sponging miR-326 as well as enhancing Hnrnpa2b1 expression.Another study also demonstrated that PCAT6 regulated RhoA-ROCK and miR-326 signaling pathways, driving M2 polarization of macrophages in cholangiocarcinoma 41 .Collectively, the PCAT6/hsa-miR-326/KDELR1 axis represented a putative regulatory pathway, which might be another reason for the upregulation of KDELR1 expression in LUAD samples.TFs can potentially modulate the mRNA expression both directly, through transcriptional regulation, and indirectly, by affecting the activity of miRNAs that target the mRNA.To explore potential TFs targeting KDELRs, we aligned the sequences of their respective promoters and identified SPI1, EP300, and MAZ as critical TFs for KDELR regulation.These TFs may directly participate in the initiation of KDELR transcription, thereby influencing their mRNA expression levels.Previous research has implicated these TFs in cancer genesis, proliferation, invasion, and metastasis [42][43][44] .On the other hand, we employed the enrichment analysis to construct a regulatory network of 11 TFs-5 miRNAs-KDELR1/2/3 relevant to LUAD progression, encompassing.This network illustrated the interconnected regulatory relationships among TFs, miRNAs, and target genes, revealing potential cascades controlling KDELR expression in LUAD.This dual mode of action illustrated the intricate interactions among TFs, miRNAs, and KDELR mRNAs in the biological regulation of cancer.
In our study, PPI network analyses were used to predict ARFGAP1, ARF1, COPB1, ARFGAP3, and COPA as the top 5 functional interactors of KDELRs.Each of these interactors has been linked to various cancers: ARFGAP1 acts as a critical regulator of mTORC1 with potential for cancer therapeutics 45 ; downregulation of ARF1 suppresses NSCLC progression 46 ; COPB1 emerges as a significant prognostic biomarker for pan-cancer 47 ; as a target gene for androgens, ARFGAP3 promotes prostate cancer progression and metastasis 48 ; and COPA is recognized as a prognostic biomarker and drug target in cervical cancer 49 .Additionally, analysis of KDELRs by GSEA revealed gene sets that enriched in the high-expression KDELRs group encompassed "MTORC1_SIGNAL-ING", "P53_PATHWAY", and "ANGIOGENESIS" from the "HALLMARK collection".These hallmarks represent upregulated gene sets involved in cell growth, tumorigenesis, and angiogenesis, respectively 50 .Furthermore, the high-expression KDELRs group was enriched for four gene sets from the "C2 collection" that display elevated expression across multiple lung cancer cell lines [51][52][53] .And the gene sets enriched in the low-expression group of KDELRs are mainly down-regulated genes in KRAS-mutated lung cancer cell lines and animal models 54 .These GSEA results collectively demonstrate a positive correlation between high KDELR1/2/3 expression and multiple upregulated gene sets in lung cancer, as well as a negative correlation with downregulated gene sets.These associations suggested that KDELR family members may be indirectly involved in these pathways leading to tumorigenesis and development.
Numerous studies have established that the immune microenvironment of tumors significantly impacts tumor progression, recurrence, and patient prognosis 55,56 .In cancer patients, the frequency of lymphocyte infiltration in tumors independently predicts survival and lymph node metastasis [22][23][24] Our findings suggest that KDELR overexpression in LUAD may negatively regulate CD8 + T cell infiltration.CD8 + cytotoxic T lymphocytes are considered the primary immune cells for targeted cancer therapies, which kill infected or tumor cells by secreting large amounts of IFN-γ and protease granzyme B 22,57 .Thus, elevated KDELR expression in LUAD may result in reduced CD8 + T cell presence within the TIME, diminishing the host's capacity to suppress tumor growth.Moreover, for immunotherapy to be effective, adequate expression of immune checkpoints, such as PD1/PD-L1 or CTLA-4, which play a pivotal role in tumor immune escape, is required 25 .We found a potentially antagonistic relationship between KDELRs and CTLA-4 in the context of LUAD, which may hold implications for unraveling the complex immunological landscape of this malignancy and guiding the development of innovative therapeutic strategies targeting both KDELRs and immune checkpoint axes.In summary, the KDELR family appears to influence the efficacy of immunotherapy in LUAD, offering valuable insights for future investigations.
This study employs the bioinformatic approach, conducting comprehensive, multi-omics analyses to elucidate the potential roles of the KDELR family genes in lung adenocarcinoma, thereby augmenting and refining the exploration of KDELR functionalities within the realm of oncology.The investigation encompasses a broad spectrum of data, including datasets from TCGA, GEO, and independent clinical cohorts from our institution, ensuring robust validation across diverse sources.Upon comparing various types of data, KDELR1 emerged as the member with the most discernible indicators.Consequently, we used KDELR1 as an example to summarize the upstream regulatory mechanisms and downstream pathways associated with its abnormal expression, as depicted in Supplementary Fig. S4.The upstream part includes a competing endogenous RNA (ceRNA) network comprising PCAT6, hsa-miR-326, and KDELR1.It also contains the promoter hypomethylation and three key TFs-SPI1, EP300, and MAZ-alongside a broader TFs-miRNAs-target genes network encompassing 4 TFs, was implicated in modulating the dysregulation of KDELR1 expression in LUAD.The downstream part includes MTORC1_SIGNALING and P53_PATHWAY, two key pathways for tumorigenesis and disease progression obtained from GSEA analysis.KDELR1 might play an indirect role in coordinating these two oncogenic pathways.It also includes B cell memory, T cell γ-δ, and T cell CD8 + in the immune microenvironment, and overexpression of KDELR1 in LUAD negatively regulated their infiltration, which might result in an immunosuppressive environment that favored tumor growth.
Our study has several limitations.On one hand, the specific functions of KDELRs in LUAD needs further experimental validation.With this in mind, we have outlined plans for upcoming experiments, including designing and constructing shRNA expression vectors targeting the KDELR family genes, leveraging lentiviral www.nature.com/scientificreports/packaging systems to establish stable LUAD cell lines with knocked-down KDELR expression.These cell lines will then serve as models for comparative assays evaluating cellular functions such as proliferation, invasion, migration, cell cycle, and apoptosis.On the other hand, the potential oncogenic mechanisms involving KDELRs in LUAD require substantiation through additional experimentation, particularly focusing on KDELR's impact on LUAD-associated signaling pathways and immune activities.To elucidate these mechanisms, we intend to follow functional assays with assessments of changes in the expression of signaling pathway marker molecules before and after target gene knockdown in our cell line models.
To conclude, the current study suggested that the KDELRs are critical signaling molecules that are highly expressed in LUAD and positively associated with poor prognosis.Methylation, ceRNA networks, and TFs are identified as potential causes of aberrant KDELR mRNA expression in LUAD.Furthermore, KDELR family members may indirectly affect pathways driving tumorigenesis and progression.Elevated KDELR expression in LUAD is associated with reduced CD8 + T cell presence within the TIME, compromising the host's ability to suppress tumor growth.Overall, these findings may provide guidance for the development of prognostic markers and novel therapeutic strategies for LUAD.

Gene expression analysis
Utilizing the R package "TCGAbiolinks," we retrieved relevant data for LUAD from The Cancer Genome Atlas (TCGA) database.Following rigorous data preprocessing steps, including deduplication and handling of missing values, we obtained Transcriptome RNA-seq data for 571 samples along with their corresponding clinical information.Among these data, we extracted expression levels of KDELR1, KDELR2, and KDELR3 in both tumor and normal tissue samples.In addition to the TCGA-based analyses, we validated our findings using an independent dataset, GSE33532, which was downloaded from the Gene Expression Omnibus (GEO) database and a group of clinical specimens from the First Hospital of Jilin University.
Considering the specific characteristics and distributional properties of the obtained expression data, appropriate statistical methods were selected to perform the analyses.Subsequently, we employed the R package "ggplot2" for data visualization, generating scatter plots and paired plots to graphically depict the expression patterns of KDELRs across different sample groups.

Protein expression analysis
We employed two distinct databases to examine protein expression profiles: the Human Protein Atlas (HPA), based on immunohistochemical techniques, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database, utilizing mass spectrometry methodologies.The HPA website (https:// www.prote inatl as.org) houses protein expression data derived from immunohistochemistry on 44 diverse normal tissues and 20 prevalent cancer types 58 .UALCAN (http:// ualcan.path.uab.edu), on the other hand, was an analytical tool grounded in the combined datasets of CPTAC and TCGA 59,60 .In the present study, these resources were leveraged to elucidate differences in the protein expression levels of KDELR1, KDELR2, and KDELR3 between LUAD and normal lung tissues.

Survival analysis
We analyzed the correlation between KDELRs mRNA expression and multiple prognostic indicators in LUAD patients using the Kaplan Meier (K-M) plotter (http:// kmplot.com/ analy sis/) 61 .The platform integrates data from the Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA), and TCGA databases, utilizing gene expression data generated via a combination of microarray and sequencing technologies.This comprehensive resource enables the evaluation of associations between the expression of all genes and patient survival across more than 30,000 samples from 21 distinct malignancies.It can also be used to assess the prognostic value of mRNAs and microRNAs in tumors 62,63 .

Amplification, deletion and mutation analysis
We analyzed the genetic alteration of KDELRs using the cBioPortal (www.cbiop ortal.org), incorporating data from ten major cancer research initiatives, including TCGA.This site provides visual and multidimensional cancer genomics data and the association between mutations and survival 64,65 .

Methylation analysis
MethSurv (https:// biit.cs.ut.ee/ meths urv/) is an online platform specifically designed for conducting survival analyses of DNA methylation biomarkers using the Cox proportional hazards model, drawing upon data from TCGA.This comprehensive dataset incorporates DNA methylation microarray data, generated using the Illumina HumanMethylation450 platform, encompassing over 10,000 samples across 34 distinct cancer types 66 .For the purposes of our investigation, we utilized MethSurv's "Gene visualization" feature to conduct cluster analysis of individual CpG sites within the target KDELR genes, presenting the results in a heatmap format.Subsequent survival analyses were then performed on these CpGs, seeking to establish correlations between methylation patterns, patient clinical characteristics, and gene subregions.Recognizing that methylation within gene promoter regions frequently functions to suppress gene transcription 28 , we further employed UALCAN (http:// ualcan.path.uab.edu) to comparatively assess methylation levels in the promoter regions of KDELR genes between LUAD and normal tissues.

Figure 1 .
Figure 1.Flowchart of construction and analysis.

Figure 3 .
Figure 3. Prognostic value of mRNA expression of KDELRs in LUAD.(A-C) OS based on "gene chip" database.(D-F) OS based on "RNA seq" database.(G-I) FP based on "gene chip" database.(J-L) RFS based on "RNA seq" database.